FIGURE 3.
Analysis of the determinants of Bok binding and molecular modeling. A, 3F-Bok constructs used to define the region that binds to IP3Rs. Shown are the N-terminal triple FLAG tag (gray box), the position of the BH1–4 domains (1–3, 14), the TM region, and the sequence of the mouse Bok BH4 domain (LGREYV), together with the mutants used to resolve the binding site. The anti-Bok epitope, residues 19–32 (4, 7), is indicated by the black bar. B, Bok homology model, based on Bax and Bak, showing the α-helical region (bright green, residues 24–42) that contains the BH4 domain (bright pink), and also the locations of the BH3 (yellow), BH1 (cyan), BH2 (blue) and TM (light pink) domains. The model starts at Pro22 and ends at Lys202 of Bok. C, models of the residue 24–45 region of BokWT and BokL34G after MD simulation for 10 ns. The positions of the BH4 domain (bright pink) and truncation points used in deletion mutants (arrows) are indicated. D, HEK 293T cells were transfected with cDNAs encoding 3F-Bok constructs and IP3R1HA, were harvested 24 h later, lysates were incubated without or with rabbit anti-HA, and IPs and input lysates were subjected to SDS-PAGE and probed for IP3R1HA and the 3F-Bok constructs.